In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [Ne-(4-amino-2-hydroxybutyl)lysine], and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We demonstrated that hypusine modification is essential for the activity of eIF-5A and for mammalian cell proliferation. We purified deoxyhypusine synthase (DHS), cloned its cDNA and extensively characterized the structural and catalytic properties of this first step enzyme. We developed potent inhibitors of DHS that exert strong antiproliferative effects in mammalian cells, including a panel of human cancer cell lines. In 2005, we have succeeded in the molecular cloning and expression of the second step enzyme, deoxyhypusine hydroxylase (DOHH). In the past reporting period, we have made a major progress in the characterization of the structure and mechanism of DOHH. The availability of the DOHH clone and protein has enabled us to overexpress fully modified eIF5A in mammalian cells and to make new approaches in the eIF5A functional studies.? ? Cloning, Expression and purification of the recombinant yeast and human DOHH. ? We over-expressed human and S. cerevisiae DOHH as GST-fusion proteins in E. coli BL23(DE3) cells using the bacterial expression vector, pGEX-4T-3. The GST-fusion enzymes were purified on GSH-Sepharose and free DOHH enzymes were purified by thrombin cleavage of the GSH-resin bound GST-DOHH. The human enzyme is a smaller protein (32 kDa protein with 302 amino acids) than the S. cerevisiae enzyme (36 kDa protein with 325 amino acids). The purified yeast and human enzymes showed comparable activities in the conversion of the deoxyhypusine-containing eIF5A to the mature hypusine-containing eIF5A. These findings confirm that the cloned yeast and human genes indeed encode DOHH enzymes.? ? DOHH is a novel HEAT-repeat-containing metalloenzyme with a structure distinct from other protein hydroxylases ? Examination of the DOHH sequence alignment reveals that it is a super helical protein containing eight tandem alpha helical hairpins (HEAT repeats). DOHH is a dyad of symmetrical N-terminal and C-terminal halves, each half consisting of four HEAT repeats, connected by a variable insert. The N-terminal half or C-terminal half was inactive alone and in mixtures, an indication that DOHH does not contain two independent active sites, and that the two symmetrical halves form one active site. DOHH contains four distinctive, strictly conserved HE (histidine-glutamate) motifs, potential metal coordination site. The DOHH structural model predicts that metal-chelating sites lie in the interior of the concave structure formed by two toroid arms. In order to validate the proposed HEAT repeat structure, CD spectral analysis was performed for the purified human DOHH. It revealed 77 % helical content, close to the value (76-78%) calculated from the predicted structure. ? The predicted super helical structure of DOHH is entirely different from the beta jellyroll structure (termed the double stranded beta helix (DSBH)) of the majority of other protein hydroxylases, e.g. the Fe(II)-and 2-oxoacid-dependent dioxygenases, prolyl or lysyl hydroxylases. ? ? Site-directed mutagenesis of DOHH and identification of amino acid residues critical for iron binding and catalysis ? In order to validate the proposed metal binding sites of DOHH, we have generated mutant enzymes with alanine substitutions of the conserved His and Glu residues. Eight single mutants, H56A, E57A, H89A, E90A, H207A, E208A, H240A, and E241A were totally devoid of DOHH activity, indicating their critical role in DOHH catalysis. Of these, six (H56A, H89A, E90A, H207A, H240A and E241A) had markedly reduced iron content, indicating that the six conserved His or Glu residues are involved in the coordination of iron at the acctive site of DOHH. Consistent with the total lack of activity of N-terminal or C-terminal half of DOHH, this finding indicates that both the iron binding and catalysis are coordinated at one active center composed of the four His-Glu motifs. The two conserved residues of His-Glu motifs, E57 and E208 that are not important for iron binding, are required for the binding of substrate protein.? ? ? DOHH is a Fe(II)-dependent enzyme with a di-iron active center: a reaction mechanism distinct from other protein hydroxylases. ? DOHH holoenzyme was separated from the metal-free, inactive apoenzyme by gel filtration and by native gel electrophoresis, since the hydrodynamic size of the apoenzyme is larger than the iron-containing holoenzyme. The stoichiometry of iron in the holoenzyme fraction was determined to be close to two mol of iron per mol of DOHH. Of various metals tested only Fe(II), but not Fe(III), stimulated the activity of the apoenzyme, indicating that Fe(II) is the catalytic metal for DOHH. The iron to DOHH stoichiometry and dependence of the iron binding and DOHH activity on each of the four conserved His-Glu motifs suggest that DOHH monomer has one dinuclear iron-containing active center, like other non-heme iron-dependent monooxygenases, such as ribonucleotide reductase and methane momooxygenase. Furthermore, addition of 2-oxoglutarate did not enhance DOHH activity of the human recombinant enzyme suggesting that its reaction mechanism is distinct from that of other 2-oxoacid and Fe(II)-dependent dioxygenases, such as prolyl 4-hydroxylase or lysyl hydroxylases. ? ? Co-transfection with vectors encoding eIF5A, DHS and DOHH is required for overproduction of hypusine-containing, active eIF5A in mammalian cells. ? In order to determine whether the cloned human DOHH gene is functional in cultured human cells, we expressed the human DOHH ORF in combination with human eIF5A-1 and DHS ORFs using mammalian expression vectors. 293T cells were transfected with p3XFLAG-CMV-7.1/heIF5A-1 alone or in combination with pCEFL/hDHS and pCEFL/hDOHH. Western blotting with eIF5A-1 antibody showed over-expression of the FLAG-eIF5A-1 in all samples transfected with p3XFLAG-CMV-7.1/heIF5A-1. However, no labeling of the FLAG-eIF5A-1 was detectable in cells transfected with FLAG-eIF5A-1 vector alone, indicating that in this case, the FLAG-eIF5A-1 protein largely exists as the unmodified precursor. Only upon co-transfection with the modification enzyme DHS, enhanced labeling of FLAG-eIF5A-1 was manifest. The labeled component of Flag-eIF5A-1in this case was deoxyhypusine, but not hypusine, indicating that DOHH was limitting. Labeling of Flag-eIF5A-1 as the hypusine form was observed only after co-transfection of FLAG-eIF5A-1 vector with both DHS and DOHH vectors. These data demonstrate that the cloned human DOHH gene expresses functional DOHH activity in mammalian cells and that co-expression of all three proteins, eIF5A, DHS and DOHH is required for overproduction of fully modified eIF5A in mammalian cells.? ? Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells? Of the two human eIF5A genes reported, normally only one isoform (eIF5A-1) is expressed in most human cell lines. eIF5A-1 and eIF5A-2 genes are differentially expressed in mammalian cells. Whereas eIF5A-1 mRNA and protein are constitutively expressed in all human cells, eIF5A-2 mRNAs (0.6-5.5 kb) are expressed in a tissue-specific manner. eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high over-expression of eIF5A-2 mRNAs. The multiple forms of eIF5A-2 mRNA, with varying lengths of the 3'-UTR were identified as products of one gene resulting from the use of different polyadenylation signals (AATAAA) in the 3'-UTR.
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